A long term goal of our research is pursuing new understanding of molecular functions in health and disease via development of label-free microscopy. In this R21 application, we develop a new method termed vibrational photoacoustic (VPA) microscopy for 3-D vibrational imaging of tissues with a field of view and a penetration depth both in the mm scale. Our method is based on excitation of molecular overtone vibration and acoustic detection of the resultant pressure waves in the tissue. Our approach is significant in the following aspects: (i) Overtone excitation provides chemical bond selectivity and spectroscopic information in a label-free manner. (ii) Acoustic detection eliminates the tissue scattering problem encountered in near-infrared spectroscopy and enables depth-resolved signal collection in one scan. (iii) Our method provides a tissue penetration depth of a few mm, which is not accessible with existing vibrational microscopies. By excitation of the second overtone of the C-H bond stretch around 8300 cm-1, where blood interference is minimal, we have demonstrated preliminary VPA imaging of tissue phantoms and atherosclerotic plaques in arteries with a penetration depth in mm scale. These results show the great potential of developing VPA microscopy and endoscopy for label-free molecular imaging and spectroscopic analysis of lipid-related disorders in live animals and eventually in patients. The two specific aims are (1) developing high-speed VPA microscopy for in vivo molecular imaging and quantitative analysis and (2) developing VPA spectroscopy and endoscopy for in situ, depth resolved characterization of atherosclerotic plaques. Successful development of VPA microscopy should provide a new platform enabling label-free molecular imaging and spectroscopic analysis of biological specimen ex vivo and in vivo. Towards diagnosis of diseases such as atherosclerosis, VPA spectroscopy aided with principal component analysis should allow determination of lesion stages owing to the capability of identifying different pathophysiological compositions throughout deep tissues. Furthermore, our endoscopy development is expected to push the VPA method towards intravital imaging of plaques. PUBLIC HEALTH RELEVANCE: (provided by applicant): Intravital imaging of vulnerable plaques in cardiovascular diseases is difficult due to limited spatial resolution and/or chemical selectivity of current imaging tools. We develop a new method termed vibrational photoacoustic microscopy for 3-D vibrational imaging of tissues with a field of view and a penetration depth both in the mm scale. Successful development of an intravascular vibrational photoacoustic probe holds the potential of detecting vulnerable plaques in a label free manner.